A major focus of my laboratory is to understand how cytoskeletal polymers help a collection of macromolecules work together to establish a common identity: to become a living cell. Grants to my laboratory from NIGMS have funded work on several basic cell biological processes, in both eukaryotes and prokaryotes, including: (i) the assembly and function of force-generating `lamellipodial' actin networks that drive membrane movements in eukaryotic cells (GM061010 and an NDC Roadmap Grant); (ii) the actin nucleating activity and biological function of Spire-family proteins (GM075287); (iii) the architecture and function of actin-based structures in the nucleus (GM061010); and (iv) DNA segregation in bacterial cells, driven by assembly of cytoskeletal polymers (GM095263 and GM079556). A MIRA grant funding all these projects would enable us redirect energy previously devoted to maintaining multiple R0-1 grants into doing more original work. To understand how molecular properties govern the architecture and function of living cells, we perform quantitative studies at multiple size scales: (i) single-molecule and bulk biochemical studies of cytoskeletal components; (ii) biophysical and microscopical studies of complex cellular structures reconstituted in vitro; and (iii) cell biological and high-resolution microscopy studies of cytoskeletal systems in living cells. Broadly speaking, the ongoing work that would be supported by this grant can be divided into four parts: 1. Studies of prokaryotic cytoskeletal systems. Prokaryotic genomes encode more than forty classes of actin-like proteins (ALPs). We have studied the assembly and function of three eubacterial ALPs (ParM, AlfA, and Alp7A) and we are currently working to understand the role of ALPs in archaea. 2. Studies of dendritic actin networks assembled by: the Arp2/3 complex, WASP/WAVE-family proteins, Ena/VASP, formins, capping protein, cofilin, and profilin. In addition to powering some types of cell locomotion, this `dendritic network motor' also contributes to phagocytosis, endocytosis, movement of intracellular pathogens, and healing of membrane ruptures. Among other things, we are currently working to understand the mechanisms by which these networks generate and adapt to mechanical forces. 3. Studies of fast amoeboid migration. We combine `Evolutionary Cell Biology' approaches with 3D Bessel Beam microscopy to understand how cells generate complex membrane dynamics and then harness them for rapid movement. Briefly, we employ comparative genomics and cell biological studies of widely divergent (non-model) organisms to uncover molecular and biophysical mechanisms of cell movement. 4. Studies of the assembly and function of actin filaments in eukaryotic nuclei. We recently discovered nuclear actin filaments created by Fmn2 and Spire-family molecules in response to DNA damage. These filaments contribute to rapid clearance of double-strand DNA breaks (Belin and Mullins, submitted) and we are working to understand their functions and to work out the signaling pathways that create them.